Characteristic distribution and molecular properties of normal cellular prion protein in human endocrine and exocrine tissues

Prion disease is an infectious and fatal neurodegenerative disease. Human prion disease autopsy studies have revealed abnormal prion protein (PrPSc) deposits in the central nervous system and systemic organs. In deer, chronic wasting disease has also become a global problem, with PrPSc in saliva and feces. Therefore, understanding normal cellular prion proteins (PrPc) characteristics in human systemic organs is important since they could be a PrPSc source. This study used western blotting and immunohistochemistry to investigate endocrine and exocrine tissues, such as the human pituitary, adrenal, submandibular glands and the pancreas. All tissues had 30–40 kDa PrP signals, which is a slightly higher molecular weight than normal brain tissue. Most cytoplasmic PrP-positive adenohypophyseal cells were immunopositive for nuclear pituitary-specific positive transcription factor 1. The adrenal medulla and islet cells of the pancreas were PrP-positive and colocalized with chromogranin A. The duct epithelium in the submandibular gland and pancreas were immunopositive for PrP. This study reports the characteristic molecular properties and detailed tissue localization of PrPc in endocrine and exocrine tissues, which is important for infection control and diagnosis.


Results
Molecular characterization of PrP. The frontal cortex samples had unglycosylated, monoglycosylated, and diglycosylated PrP signals, ranging from 25 to 38 kDa using the 3F4 antibody (Fig. 1A). The PrP signal intensity varied per tissue depending on the PrP antibody and the case ( Table 2). The pituitary samples had a PrP smear band around 30-40 kDa (Fig. 1A), and the adrenal gland samples had PrP smear bands around 16-20 kDa in addition to those around 30-40 kDa. The pancreatic PrP signal pattern was nearly identical to the adrenal gland pattern, with ~ 16-20 kDa and ~ 30-40 kDa smear bands (Fig. 1C). However, the signal intensity  www.nature.com/scientificreports/ differed between cases, and case 4 was challenging to detect (Fig. 1C, asterisk). The submandibular gland samples had PrP signal patterns similar to those of the adrenal gland and pancreas with PrP signals of ~ 16-20 kDa and ~ 30-40 kDa (Fig. 1D). However, it was difficult to detect the PrP signal in case 1 (Fig. 1D, asterisk). In the case 6 and 7, western blot analysis with 3F4 antibody and EP1802Y antibody showed PrP signals almost similar to the case 1-5 (  Fig. S4). Next, another PrP antibody (EP1802Y) was used to re-examine the PrP signal patterns in these membranes, and the ~ 16-20 kDa and ~ 30-40 kDa smear bands were observed with the 3F4 antibody were also present with the EP1802Y antibody (Suppl. Fig. S5A-D).
Immunohistochemistry for TBX19 revealed nuclear immunopositivity in some adenohypophyseal cells (Fig. 5I). Double immunofluorescence of PrP (3F4) and TBX19 ( Fig. 5J-L) showed only a small number of cytoplasmic PrP-positive adenohypophyseal cells with nuclear TBX19 positivity (9.6%, 5/52). Immunohistochemistry for SF-1 revealed nuclear staining in the adenohypophyseal cells (Fig. 5M, arrowheads). However, double immunofluorescence of SF-1 and EP1802Y was difficult because of the different antigen activation methods. Therefore, we used a mirror image in the adjacent section. In the mirror image for SF-1 and PrP (3F4), most nuclear SF-1 positive adenohypophyseal cells were negative for cytoplasmic PrP (Fig. 5N, arrowheads). CgA immunopositivity was observed in most adenohypophyseal cells and was strongly expressed in some (Fig. 5O, arrowhead). Furthermore, double immunofluorescence of CgA and PrP (EP1802Y) revealed colocalization in most adenohypophyseal cells. However, highly CgA-expressing cells showed no cytoplasmic PrP (Fig. 5P, arrowhead).

Discussion
This study demonstrated the characteristic molecular properties of PrP in several human endocrine and exocrine tissues, such as the pituitary, adrenal, and submandibular glands and the pancreas, which have different characteristics than the brain. Additionally, we detail the histological localization of PrP in these tissues. Western blotting showed PrP smear bands ranging from 30 to 40 kDa in the pituitary gland, similar to previous reports 5 . These molecular weights are higher than those for the brain, and our previous study showed that this was due to excessive glycosylation. In western blot analysis from FFPE samples, PrP signals were observed www.nature.com/scientificreports/ in the adrenal, pancreas, and submandibular glands at ~ 30-40 kDa and ~ 16-20 kDa. However, western blot analysis from frozen samples showed no ~ 16-20 kDa signals. Therefore, the ~ 16-20 kDa signals seen in FFPE samples were likely to be an artifact that occurred in the fixation and embedding procedures the FFPE samples. The ~ 30-40 kDa PrP smear signal was similar to that of the pituitary gland. Both in the FFPE samples and frozen samples, after deglycosylation treatment with PNGaseF, the ~ 30-40 kDa PrP smear signal was converged to a single band at the molecular weight of about 25 kDa which corresponds to a nonglycosylated PrP signal. Therefore, PrP of organs such as the pancreas, adrenal gland, and submandibular gland also undergoes excessive glycosylation compared to the cerebrum. Unlike PrP in the cerebrum, PrP in endocrine/exocrine tissues may be difficult to separate by excessive glycosylation like pituitary PrP. Pit-1 activates transcription of the GH, prolactin, and beta-thyroid stimulating hormone genes 19 , and we found that most cytoplasmic PrP c positive adenohypophyseal cells were immunopositive for nuclear Pit-1. Conversely, cytoplasmic PrP c positive adenohypophyseal cells were essentially negative for SF-1 and TBX19. These findings suggest that the expression of PrP c in adenohypophyseal cells tents to occur in differentiated cells downstream from somatotroph stem cells rather than corticotroph cells and gonadotroph cells. A previous report showed that cytoplasmic PrP c positive adenohypophyseal cells are frequently positive for GH or prolactin 5 , and our results agree. However, there are no reports that suggest a direct association between PrP and Pit-1, and it is unclear when PrP c is highly expressed during cell differentiation by Pit-1.
In the adrenal gland, PrP c immunoreactivity was observed in the adrenal medulla. The adrenal medulla is derived from the neural crest, and PrP c is as abundant as in the central nervous system. For example, PrP Sc was detected in the adrenal medulla in cattle affected with bovine spongiform encephalopathy and scrapie-infected sheep 20,21 . In humans, we previously reported PrP Sc aggregation in the adrenal medulla of sCJD patients and enhanced seeding activity on real-time quaking-induced conversion 22 . Our results suggested that PrP c in the adrenal medulla may be the source of PrP Sc .
In the pancreas, islet cells have cytoplasmic PrP. In rats, PrP c is abundant in islet cells and is involved in regulating blood glucose homeostasis 23 . In addition, PrP c primarily expresses in pancreatic β cells in humans and may contribute to insulin resistance 24,25 . CgA is a neuroendocrine protein that is found in the adrenal medulla and sympathetic nerve endings and is localized to nerve cells and secretory vesicles of endocrine cells 26 . CgA function as a low-affinity Ca2+ binding protein in secretory granules of neuroendocrine cells. In addition, CgA are also observed in the lumen of the ER of many cell types and play modulatory role in the control of Ca2+ release   1  3  3  3  1  1  2  0  0  1  0  0  1   2  3  3  3  3  2  3  0  0  2  2  1  3   3 n.a n.a n.a 1 1 1  1  1  1  1  2  3   4  3  3  3  2  2  2  1  1  2  1  2  3   5  1  2  3  1  2  2  1  1  2  1  2  3   6  3  3  3  3  1  2  1  0  1 n.a n.a n.a  www.nature.com/scientificreports/ PrP immunopositivity was detected in the ductal epithelium of the submandibular gland and the pancreas. There have been no reports of PrP expression in the ductal epithelium of animals or humans. CWD is a fatal neurodegenerative prion disease that affects species of the Cervidae family and is now a widespread global problem in the United States, Canada, and Europe 12,28,29 . Saliva and feces containing PrP Sc are likely sources of infection 28,29 . Our results suggest that in CWD animals, PrP Sc may be secreted from the ductal epithelium in saliva and from the pancreas through the gastrointestinal tract to feces.
This study detected PrP c in human endocrine and exocrine tissues using western blotting and clarified the histological details. PrP c is an element of the crinophagy mechanism (an autophagy mechanism that processes excess hormones and neurotransmitters) in secretory cells. Furthermore, PrP c is involved in the secretory pathway of secretory granules 8 . The immunohistochemistry and western blotting detection sensitivities for PrP c varied between cases. This may be due to different endocrine and exocrine function states per case during the period before death. The immunohistochemistry detection sensitivities also differed depending on the PrP antibody, which may be related to antigen exposure owing to the three-dimensional structure of PrP c . This study includes several limitations. Frozen samples of endocrine and exocrine tissue were not sufficiently collected at autopsy, therefore, analysis of western blots was mainly performed from FFPE samples. It is undeniable that modification of the sample during the FFPE manufacturing process can affect the results of western blot. However, western blot results from FFPE of the brain and pituitary gland were similar to those of frozen samples. In addition, since it is a biochemical result of the tissue adjacent to the specimen used for histological observation, we think that it is also a very suitable method for examining the tissue localization of PrP c . Therefore, we believe that the characteristic signals found in western blots of the adrenal glands, pancreas and submandibular glands are reliable.

Conclusion
In this study, we demonstrated PrP c in human endocrine/exocrine tissues including pituitary, adrenal, submandibular glands and the pancreas. Recently, in prion diseases, PrP Sc deposits have been observed not only in the central nervous system but also in systemic organs including endocrine tissues. Our results may indicate a source of PrP Sc in systemic organs. At the same time, they can also be a source of infection. The detailed function of PrP c in endocrine and exocrine tissues is still unknown. Therefore, clarifying the relationship between PrP c and secretory function, and monitoring endocrine and exocrine function in prion disease patients are important for infection control and prompt diagnosis.

Data availability
All data supporting the findings of this study are available within the article and its supplementary files.